Several publications and patent documents are referenced in this application in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications and documents is incorporated by reference herein.
The ability of mammalian cells to survive chromosomal damage depends on the coordinated activity of DNA response pathways that halt cell cycle progression and facilitate DNA repair prior to DNA replication or mitosis. The Ataxia Telangiectasia Mutated (ATM) kinase plays a crucial function as a transducer of the DNA damage signal [reviewed in (Abraham. (2001) Genes Dev 15, 2177-2196; Kastan and Lim. (2000) Nat Rev Mol Cell Biol 1, 179-186; Shiloh. (2003) Nat Rev Cancer 3, 155-168; Zhou and Elledge. (2000) Nature 408, 433-439].Following the generation of double-strand breaks (DSBs) by IR or clastogens, inactive ATM kinase dimers are converted into active monomers through autophosphorylation on serine 1981 [Bakkenist and Kastan. (2003) Nature 421, 499-506]. ATM has a number of direct targets, including Nbs1, Chk2, Mdm2, 53BP1, BRCA1, Rad17, Smc1, FANCD2, and H2AX [reviewed in (D'Andrea and Grompe. (2003) Nat Rev Cancer 3, 23-34; Kastan and Lim. (2000) supra; Shiloh. (2003) supra]. The ATM- and Rad3-related kinase, ATR, responds to replication stress and UV damage as well as to DSBs [reviewed in (Abraham. (2001) supra]. ATR requires an interacting partner, ATRIP, and the loading of RPA on single-stranded DNA for its activation [Cortez et al. (2001) Science 294, 1713-1716; Zou and Elledge. (2003) Science 300, 1542-1548]. There is considerable redundancy in the ATM and ATR signaling pathways in part because most effectors can be phosphorylated by both kinases.
Many ATM targets and other proteins involved in the DNA damage response physically accumulate at or near DNA lesions [Haaf et al. (1995). Proc Natl Acad Sci USA 92, 2298-2302; reviewed in (Petrini and Stracker. (2003) supra). As was first shown by indirect immunofluorescence (IF) for the Mre11 complex [Nelms et al. (1998) Science 280, 590-592] large multimeric complexes, termed ionizing radiation induced foci (IRIF) are formed at sites of DNA damage. IRIFs contain numerous additional factors, including ATM, ATR, ATRIP, β-H2AX, Rad17, Chk1, 53BP1, and BRCA1.
A hallmark of AT cells is their diminished ability to survive ionizing radiation (IR) or other genotoxic treatments that create double-strand breaks [Taylor et al. (1975) Nature 258, 427-429; reviewed in (Shiloh, 2003, supra]. The ATM-dependent DNA damage response can block progression through the cell cycle before, during, and after DNA replication. DNA damage-induced cell cycle arrest in late G1/early S phase involves ATM-dependent activation of p53, a process that is mediated by Chk2 and Mdm2 [Hirao et al. (2000) Science 287, 1824-1827; Kastan et al. (1992) Cell 71, 587-597; Maya et al. (2001) Genes Dev 15, 1067-1077]; reviewed in Abraham (2001) supra]. The intra-S phase checkpoint involves ATM-mediated phosphorylation of Nbs1, Smc1, and BRCA1 [Gatei et al. (2000) Nat Genet 25, 115-119; Kim et al. (2002) Genes Dev 16, 560-570; Lim et al. (2000) Nature 404, 613-617; Wu et al. (2000) Nature 405, 477-482; Xu et al. (2001) Mol Cell Biol 21, 3445-3450; Xu et al. (2002b) Cancer Res 62, 4588-4591; Yazdi et al. (2002) Genes Dev 16, 571-582; Zhao et al. (2000) Nature 405, 473-477]. ATM controls a second parallel intra-S phase pathway that is dependent on Chk2-mediated phosphorylation of Cdc25A, resulting in degradation of Cdc25A and reduced Cdk2 activity [Falck et al. (2001) Nature 410, 842-847; Falck et al. (2002) Nat Genet 30, 290-294]. When the intra-S phase checkpoint is compromised, radioresistant DNA synthesis (RDS) takes place, a second hallmark of AT cells. ATM is also important for the G2/M arrest after IR, a pathway that involves phosphorylation of BRCA1 and inhibition of Cdc25C by Chk2 ATM [Matsuoka et al. (1998) Science 282, 1893-1897; Xu et al. (2001) Mol Cell Biol 21, 3445-3450].
The ATM gene is an example of a complex polyexonic eukaryotic gene that codes for a large protein product, defects in which appear as autosomal recessive mutations. Ataxia telangiectasia (AT), which presents in patients that possess mutations in both alleles of the ATM gene, is an autosomal recessive, multi-system disorder that leads to progressive neuromuscular and vascular degeneration. As indicated herein above, chromosomal breakage and rearrangement are characteristic features of AT cells, which are abnormally sensitive to ionizing radiation. This hypersensitivity is evident in homozygous recessive AT patients and heterozygous carriers, both of which genotypes/phenotypes are predisposed to the development of cancer.
Because of the severity of the disease associated with mutations in the ATM gene, accurate and early detection of an ATM mutation in a patient is critical for the care of the affected individual. Moreover, patients or families frequently request confirmation of a suspected diagnosis of AT. Definitive detection of an ATM mutation in a patient merits additional screening of the patient's family members to identify other individuals possessing the mutation in either homozygous or heterozygous form. Since carriers of ATM mutations (i.e., heterozygotes with one normal gene) may also display an increased risk for cancer, particularly breast cancer, testing for such mutations is imperative because early detection is a critical predictive factor for improved prognosis in cancer patients. Early detection also enables the patient and healthcare providers thereof to take precautionary measures to minimize the exposure of the patient to ionizing radiation.
Improved methods for detecting polymorphisms in the ATM gene are, therefore, needed. Available techniques include restriction endonuclease fingerprinting (REF), the single-stranded conformation polymorphism (SSCP) technique, and the protein truncation test (PTT). Each of these methods, however, suffers from a variety of drawbacks. In general, the methodology used to screen for mutations biases the types of mutations that can be found. The PTT, for example, cannot detect mutations occurring in non-coding regions such as control elements. Thus, a need for improved methods for detecting mutations and polymorphisms in complex polyexonic eukaryotic structural genes such as ATM exists.